Functional Mapping of the Nucleoprotein of Ebola Virus

Abstract

) previously demonstrated that NP, together with the minor matrix protein VP24 and polymerase cofactor VP35, is necessary and sufficient for the formation of nucleocapsid-like structures that are morphologically indistinguishable from those seen in Ebola virus-infected cells. They further showed that NP is O glycosylated and sialylated and that these modifications are important for interaction between NP and VP35. However, little is known about the structure-function relationship of Ebola virus NP. Here, we examined the glycosylation of Ebola virus NP and further investigated its properties by generating deletion mutants to define the region(s) involved in NP-NP interaction (self-assembly), in the formation of nucleocapsid-like structures, and in the replication of the viral genome. We were unable to identify the types of glycosylation and sialylation, although we did confirm that Ebola virus NP was glycosylated. We also determined that the region from amino acids 1 to 450 is important for NP-NP interaction (self-assembly). We further demonstrated that these amino-terminal 450 residues and the following 150 residues are required for the formation of nucleocapsid-like structures and for viral genome replication. These data advance our understanding of the functional region(s) of Ebola virus NP, which in turn should improve our knowledge of the Ebola virus life cycle and its extreme pathogenicity.Ebola and Marburg viruses are filamentous, enveloped, nonsegmented, negative-stranded RNA viruses of the family Filoviridae in the order Mononegavirales, which also includes the Paramyxoviridae, Rhabdoviridae, and Bornaviridae (7, 32). Despite the fact that the severe hemorrhagic fever caused by Ebola virus is associated with extremely high mortality rates in human and nonhuman primates, an effective vaccine or antiviral drugs have yet to be developed.Ebola virus particles consist of at least seven structural proteins encoded by a single-stranded negative-sense RNA genome. Four of these proteins-nucleoprotein (NP), VP35, VP30, and the RNA-dependent RNA polymerase (L)-are components of the ribonucleoprotein complex that is responsible for the transcription and replication of the viral genome (26). Three others-glycoprotein (GP), VP40, and VP24-are membrane-associated proteins (12, 32), and VP24 is also thought to be involved in nucleocapsid formation (15).The NP of Ebola virus is the largest (739 amino acid residues) nucleoprotein of the nonsegmented negative-stranded RNA viruses and can be divided into a hydrophobic N-terminal half (approximately 350 amino acids) and a hydrophilic C-terminal half (33). Huang et al. (15) showed that Ebola virus NP is O glycosylated and sialylated and that these modifications are required for its interaction with VP35, which suggests that the modifications are important for viral genome replication. However, the functional region(s) of Ebola virus NP has not been characterized. The nucleoproteins (NP/N) of nonsegmented negative-stranded RNA viruses, including Marburg virus NP, are known to...

Filovirus Replication and Transcription

“…According to this model, NP forms helical tubes which differ from nucleocapsid-like structures. VP35 and VP24 interact with the pre-formed NP-derived helices, resulting in the formation of nucleocapsid-like structures [36]. Both transport of the nucleocapsids to the plasma membrane and incorporation into virions are mediated by the interaction of NP with VP40 [23,37].…”

Ebolavirus Classification Based on Natural Vectors

“…The NP of ebolavirus is necessary and sufficient for the formation of nucleocapsid-like structures in a mammalian expression system. It is also responsible for the NP-NP interaction and replication of the viral genome (Watanabe et al, 2006). Xu (2014) showed that VP24 binds to the karyopherin alpha nuclear transporters and blocks the nuclear accumulation of STAT1, a very important component of the immune system (Xu et al, 2014).…”

Predictive and comparative analysis of Ebolavirus proteins

“…NP encapsidates the genome and protects it from nucleases. 17,18 VP30 is a transcription anti-terminator 19,20 and regulates the switch between transcription and replication. 21,22 VP35 acts as a cofactor of the polymerase, 23,24 and VP40 may also play a role in genome replication and transcription.…”